Method and apparatus for multi-modal imaging

ABSTRACT

A method and apparatus for imaging a subject animal. The method comprises the steps of treating the animal with an x-ray contrast agent and an imaging agent; supporting the animal in an immobilized state on a support member; acquiring an x-ray anatomical image of the animal; acquiring an optical, dark-field image of the animal; and registering the x-ray anatomical image and the optical image, whereby features of the optical image can be observed in relation to features of the anatomical image.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a Continuation of U.S. Ser. No. 12/460,010 filed Jul. 10, 2009by Leevy et al entitled METHOD AND APPARATUS FOR MULTI-MODAL IMAGING,incorporated herein in its entirety, which itself claimed priority toU.S. Provisional Patent Application Ser. No. 61/079,847 filed Jul. 11,2008 by Leevy et al and entitled APPARATUS AND METHOD FOR MULTI-MODALIMAGING, the contents of which are incorporated by reference into thisspecification.

This application is a continuation-in-part of the following commonlyassigned, copending U.S. patent applications, the contents of each ofwhich also are incorporated by reference into this specification:

U.S. Ser. No. 11/221,530, filed Sep. 9, 2005 by Vizard et al, entitledAPPARATUS AND METHOD FOR MULTI-MODAL IMAGING; and

U.S. Ser. No. 12/354,830 filed Jan. 6, 2009 by Feke et al, entitledAPPARATUS AND METHOD FOR MULTI-MODAL IMAGING.

FIELD OF THE INVENTION

The invention relates generally to the field of imaging systems, andmore particularly to the imaging of objects. More specifically, theinvention relates to an improved apparatus and method that enableanalytical imaging of objects (for example, small animals and tissue) indiffering modes, including bright-field, dark-field (e.g., luminescenceand fluorescence), and x-ray and radioactive isotopes.

BACKGROUND OF THE INVENTION

Electronic imaging systems are well known for enabling molecularimaging. An exemplary electronic imaging system 10 (shown in FIG. 1 anddiagrammatically illustrated in FIG. 2) is the Image Station 2000MMMultimodal Imaging System formerly available from the Eastman KodakCompany. System 10 includes a light source 12, an optical compartment 14which can include a mirror 16, a lens and camera system 18, and acommunication and computer control system 20 which can include a displaydevice, for example, a computer monitor 22. Camera and lens system 18can include an emission filter wheel for fluorescent imaging. Lightsource 12 can include an excitation filter selector for fluorescentexcitation or bright field color imaging. In operation, an image of anobject is captured using lens and camera system 18. System 18 convertsthe light image into an electronic image, which can be digitized. Thedigitized image can be displayed on display device 22, stored in memory,transmitted to a remote location, processed to enhance the image, usedto print a permanent copy of the image, or all of these.

SUMMARY OF THE INVENTION

An object of the present invention is to provide an improved method andapparatus for enabling analytical imaging of an object. Another objectof the present invention is to provide such a method and apparatus thatuse differing imaging modes.

These objects are given only by way of illustrative example, and suchobjects may be exemplary of one or more embodiments of the invention.Other desirable objectives and advantages inherently achieved by thedisclosed invention may occur or become apparent to those skilled in theart. The invention is defined by the appended claims.

According to one aspect of the present invention, there is provided animproved method for using an imaging system for imaging an object. Anexample of such an imaging system useful in the inventive methodincludes a support member adapted to receive the object in animmobilized state. The system also includes first means for imaging theimmobilized object in a first imaging mode to capture a first image, andsecond means for imaging the immobilized object in a second imagingmode, different from the first imaging mode, to capture a second image.The first imaging mode is selected from the group: x-ray mode and radioisotopic mode. The second imaging mode is selected from the group:bright-field mode and dark-field mode. A removable phosphor screen maybe employed when the first image is captured, but not employed when thesecond image is captured. The phosphor screen is adapted to transduceionizing radiation to visible light. The phosphor screen is adapted tobe removable without moving the immobilized object. The system canfurther include means for generating a third image comprised of thefirst and second image.

A first embodiment of the inventive method is useful for imaging asubject animal. The method includes a step of treating the animal withboth an x-ray contrast agent and an optical imaging agent, which may betargeted or non-targeted agents, or both. As used in this specificationand as understood by those skilled in the art, the terms “targetedagent” refer in general to agents that accumulate in specific tissues ororgans of the animal's body by molecular targeting, such as antibodies,peptides and the like attached to the agents. Similarly, the terms“non-targeted agents” refer in general to agents that accumulate inspecific tissues or organs by physiological processes, such as thegastrointestinal process or the renal process. The method of theinvention further may include steps of supporting the animal in animmobilized state; acquiring an x-ray anatomical image of theimmobilized animal; acquiring an optical dark-field image, such as afluorescence image or a luminescence image, of the immobilized animal;and registering the x-ray anatomical image and the optical dark-fieldimage, whereby features of the optical image can be observed in relationto features of the anatomical image. The x-ray contrast agent andoptical imaging agent may be administered simultaneously or sequentiallyto the animal. In accordance with the invention, the x-ray contrastagent may be targeted while the optical imaging agent is non-targeted;or the x-ray contrast agent may be non-targeted while the opticalimaging agent is targeted; or both agents may be targeted; or bothagents may be non-targeted. The x-ray image and the optical image may beacquired using a common, shared focal plane.

A second embodiment of the inventive method may include steps oftreating the animal with an x-ray contrast agent and an optical imagingagent, as in the first embodiment; supporting the animal in animmobilized state on a support member; providing a phosphor plateadapted to be disposed proximate the support member when capturing afirst image; with the phosphor plate disposed proximate the supportmember, imaging the immobilized animal in a first imaging mode tocapture the first image, the first imaging mode being an x-ray mode;removing the phosphor plate from proximate the support member, aftercapturing the first image and without moving the immobilized animal andthe support member; and with the phosphor plate removed from proximatethe support member, imaging the immobilized animal in a second imagingmode to capture a second image, the second imaging mode being adark-field mode. The method may include a further step of generating athird image by merging the first and second images, whereby features ofthe second image can be observed in relation to features of the firstimage. Again, the x-ray contrast agent and optical imaging agent may beadministered simultaneously or sequentially to the animal. Also, thex-ray contrast agent may be targeted while the imaging agent isuntargeted; or the x-ray contrast agent may be non-targeted while theimaging agent is targeted; or both agents may be targeted; or bothagents may be untargeted. The x-ray image and the optical image may beacquired using a common, shared focal plane.

A third embodiment of the inventive method may include steps of treatingthe animal with an x-ray contrast agent and an optical imaging agent asin the first and second embodiments; supporting the animal in animmobilized state on a support member; providing a phosphor platemovable relative to the support member, without disturbing theimmobilized animal and the support member, between a first positionwherein the phosphor plate is in optical registration with the supportmember and a second position wherein the phosphor plate is not inoptical registration with the support member; capturing an x-ray imageof the immobilized animal when the phosphor plate in disposed in thefirst position; and capturing a dark-field image of the immobilizedanimal when the phosphor plate in disposed in the second position. Themethod may include further steps of generating a third image by mergingthe first and second images, whereby features of the second image can beobserved in relation to features of the first image; and displaying,transmitting, processing, or printing, the third image. As in the firsttwo embodiments, the x-ray contrast agent and optical imaging agent maybe administered simultaneously or sequentially to the animal. Also, thex-ray contrast agent may be targeted while the imaging agent isuntargeted; or the x-ray contrast agent may be non-targeted while theimaging agent is targeted; or both agents may be targeted; or bothagents may be untargeted. The x-ray image and the optical image may beacquired using a common, shared focal plane.

A fourth embodiment of the invention concerns an apparatus for imaging asubject animal. This apparatus may include first imaging means forimaging such an animal in a first imaging mode to capture a first image,the first imaging mode being selected from the group: x-ray mode andradio isotope mode; second imaging means for imaging such an animal in asecond imaging mode that uses light from the immobilized animal tocapture a second image, the second imaging mode being selected from thegroup: bright-field imaging mode and dark-field imaging mode; and asupport stage, fixedly mounted in the apparatus, for receiving such ananimal in an immobilized state such that the animal is immobilized inthe apparatus during imaging by the first and second imaging meanswithout movement of the animal from the support stage or movement of thesupport stage between capture of the first and second images.

The fourth embodiment also may include movable phosphor plate totransduce ionizing radiation from the first imaging means to visiblelight, the phosphor plate being mounted to be moved, without moving theimmobilized animal and support stage, between a first position proximatethe support stage during capture of the first image and a secondposition not proximate the support stage during capture of the secondimage.

A fifth embodiment of the invention concerns a method for imaging asubject animal. This method may include steps of providing a fixedsupport stage; receiving the animal on the support stage in animmobilized state; imaging the immobilized animal on the support stagein a first imaging mode to capture a first image, the first imaging modebeing selected from the group: x-ray mode and radio isotope mode; andwithout moving the animal or the support stage, imaging the animal onthe support stage in a second imaging mode that uses light from theimmobilized animal to capture a second image, the second imaging modebeing selected from the group: bright-field imaging mode and dark-fieldimaging mode.

This fifth embodiment also may include steps of providing a movablephosphor plate to transduce ionizing radiation from the first imagingmeans to visible light; and moving the phosphor plate, without movingthe immobilized animal and support stage, between a first positionproximate the support stage during capture of the first image and asecond position not proximate the support stage during capture of thesecond image. This method further may include steps of treating theanimal with an x-ray contrast agent and an optical imaging agent; andregistering the first image with the second image, whereby features ofthe second image may be observed in relation to features of the firstimage.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features, and advantages of theinvention will be apparent from the following more particulardescription of the embodiments of the invention, as illustrated in theaccompanying drawings. The elements of the drawings are not necessarilyto scale relative to each other.

FIG. 1 shows a perspective view of an exemplary prior art electronicimaging system, of a type useful in accordance with the method of thepresent invention.

FIG. 2 shows a diagrammatic view of the system of FIG. 1.

FIG. 3A shows a diagrammatic side view of the imaging system of FIGS. 1and 2.

FIG. 3B shows a diagrammatic front view of the imaging system of FIG.3A.

FIG. 4 shows a perspective view of the imaging system of FIGS. 3A and3B.

FIG. 5A shows a diagrammatic side view of the sample object stage,showing the relative movement of the phosphor plate relative to thesample object stage.

FIG. 5B shows a diagrammatic side view of the sample object stage in thefirst imaging position P1 wherein the phosphor plate is disposedproximate the sample object stage.

FIG. 5C shows a diagrammatic side view of the sample object stage in thesecond imaging position P2 wherein the phosphor plate is not proximatethe sample object stage.

FIG. 6 shows an enlarged, fragmentary sectional view taken along line6-6 of FIG. 5B.

FIG. 7 shows an enlarged, fragmentary sectional view taken along line7-7 of FIG. 5C.

FIG. 8 shows a work flow diagram in accordance with the mode ofoperation of the system of FIGS. 1 to 7.

FIG. 9A shows a first image of an immobilized object in a first,fluorescence imaging mode.

FIG. 9B shows a second image of the immobilized object of FIG. 9A in asecond, x-ray imaging mode.

FIG. 9C shows an image generated by thresholding the image of FIG. 9Aand then merging that image with the image of FIG. 9B.

FIG. 10A shows a first image of an immobilized object in a first,fluorescence imaging mode.

FIG. 10B shows a second image of the immobilized object of FIG. 10A in asecond, x-ray imaging mode.

FIG. 10C shows an image generated by thresholding the image of FIG. 10Aand then merging that image with the image of FIG. 10B.

FIG. 11A shows a first image of an immobilized object in a first,fluorescence imaging mode.

FIG. 11B shows a second image of the immobilized object of FIG. 11A in asecond, x-ray imaging mode.

FIG. 11C shows an image generated by thresholding the image of FIG. 11Aand then merging that image with the image of FIG. 11B.

FIG. 12 is a diagrammatic view of a suitable phosphor plate for use withthe apparatus suitable for practice of the method of the presentinvention.

FIG. 13 is a flow diagram of a method for making a phosphor plate ofFIG. 12.

FIG. 14A shows a diagrammatic partial view of a mouse in a samplechamber on the sample object stage of the imaging system of FIGS. 3A and3B when an X-ray anatomical image, with an X-ray contrast agentproviding contrast of the gastro-intestinal tract of the subject, isacquired in accordance with the present invention.

FIG. 14B shows an x-ray anatomical image captured using the imagingsystem of FIG. 14A.

FIG. 15A shows a diagrammatic partial view of a mouse in a samplechamber on the sample object stage of the imaging system of FIGS. 3A and1B when a near-infrared fluorescence image of the gastro-intestinaltract is acquired in accordance with the present invention.

FIG. 15B shows a near-infrared fluorescence image captured using theimaging system of FIG. 15A.

FIGS. 16A and 16B respectively show an anatomical X-ray image and anear-infrared fluorescence image of the gastro-intestinal tract of asubject acquired in accordance with the method of the present invention.

FIGS. 17A, 17B and 17C respectively show an anatomical X-ray image withan X-ray contrast agent providing contrast of the kidneys of thesubject, a near-infrared fluorescence image of the kidneys of thesubject, and a co-registered image of the anatomical X-ray image and thenear-infrared fluorescence images of the kidneys of the subject,acquired in accordance with the method of the present invention.

FIG. 18 shows a workflow diagram in accordance with a method of thepresent invention.

FIG. 19A shows a diagrammatic partial view of a mouse in a samplechamber on the sample object stage of the imaging system of FIGS. 3A and3B when an X-ray anatomical image, with an X-ray contrast agentproviding contrast of the kidneys of the subject, is acquired inaccordance with the present invention.

FIG. 19B shows an x-ray anatomical image captured using the system ofFIG. 19A.

FIG. 20A shows a diagrammatic partial view of a mouse in a samplechamber on the sample object stage of the imaging system of FIGS. 3A and1B when a near-infrared fluorescence image of the kidneys is acquired inaccordance with the present invention.

FIG. 20B shows a near-infrared fluorescence image captured using thesystem of FIG. 20A.

FIG. 21 shows an anatomical X-ray image of a mouse in a sample chamberon the sample object stage of the imaging system of FIGS. 3A and 3B.

FIG. 22 shows a graph of the excess x-ray density in the medullaryregions of the kidneys conferred by the X-ray contrast agent of FIG. 19vs. the injected volume of the X-ray contrast agent.

DETAILED DESCRIPTION OF THE INVENTION

The following is a detailed description of the preferred embodiments ofthe invention, reference being made to the drawings in which the samereference numerals identify the same elements of structure in each ofthe several figures.

In the complex pharmaceutical analyses of small objects or subjects suchas small animals and tissue samples, images are particularly enhanced byusing different in-vivo imaging modalities. Using the known or currentpractices of bright-field, dark-field and radiographic imaging for theanalysis of small objects or subjects such as a mouse can be expensiveand may not provide the precision of co-registered images that isdesired.

Using the method and apparatus of the present invention, preciselyco-registered images can be obtained using x-ray and fluorescent,luminescent, or radioactive isotopic probes, individually or incombination, within an object such as a live animal or a tissue sample.The images can be localized and multiple images can be accuratelyoverlaid onto the simple bright-field reflected image or anatomicalx-ray image of the same animal within minutes of animal immobilization.

The method and apparatus of the present invention use the same imagingsystem to capture images using different modes of imaging, therebyenabling or simplifying multi-modal imaging. In addition, relativemovement of imaging probes can be kinetically resolved over the timeperiod that the animal is effectively immobilized, which can be tens ofminutes. Alternatively, the same animal may be subject to repeatedcomplete image analysis over a period of days or weeks required toassure completion of a pharmaceutical study, with the assurance that theprecise anatomical frame of reference (particularly, the x-ray image)may be readily reproduced upon repositioning the object animal. Themethod of the present invention can be applied to other objects orcomplex systems, or both, subject to simple planar imagingmethodologies. More particularly, using the imaging method of thepresent invention, an immobilized object can be imaged in severalimaging modes without changing or moving the immobilized object. Theseacquired multi-modal images can then be merged to provide aco-registered image for analysis.

Imaging modes supported by the method of the present invention include:x-ray imaging, bright-field imaging, dark-field imaging (includingluminescence imaging, fluorescence imaging) and radioactive isotopeimaging. Images acquired in these modes can be merged in variouscombinations for analysis. For example, an x-ray image of the object canbe merged with a near-infrared fluorescence image of the object toprovide a new image for analysis.

The use of molecular imaging has engendered a need to co-registerfluorescent and luminescent, or radioactive isotope signals withanatomical features of the animal or specimen. The type of imagingsystem particularly useful for the method and apparatus of the presentinvention utilizes a low energy X-ray source and phosphor screen tosupplement the optical imaging modalities. An X-ray image provides aconvenient anatomical map of musculoskeletal features, and is anorthogonal imaging modality that will not pollute optical signalsemanating from the subject. While the skeleton provides the framework tospatially assign organs, X-ray contrast agents are used in the method ofthe invention to provide an effective means to delineate soft tissuesthat typically give poor contrast by X-ray. For example, many optical orradioisotopic signals emanate from soft tissues like the kidneys, liver,and gastrointestinal tract during circulation and clearance. Thus, it isparticularly important to identify and delineate these organ structuresin an X-ray to provide anatomical co-registration of these signals.

An embodiment of the apparatus of the present invention, useful topractice the method of the invention, is now described with reference toFIGS. 3A, 3B, and 4. Imaging system 100 includes light source 12,optical compartment 14, a lens and camera system 18, and communicationand computer control system 20 which can include computer monitor 22. Asbest shown in FIG. 3A, imaging system 100 includes an x-ray source 102and a sample object stage or support member 104. An immobilized object,such as a mouse, is received on and supported by sample object stage 104during use of system 100. Imaging system 100 further comprisesepi-illumination, for example, using fiber optics 106, which directsconditioned light (of appropriate wavelength and divergence) towardsample object stage 104 to provide bright-field or fluorescent imaging.Sample object stage or support member 104 is disposed within a sampleenvironment 108, which allows access to the object being imaged.Preferably, sample environment 108 is light-tight and fitted withlight-locked gas ports (not illustrated) for environmental control. Suchenvironmental control might be desirable for controlled x-ray imaging orfor support of particular specimens. Environmental control enablespractical x-ray contrast below 8 Kev (air absorption) and aids in lifesupport for biological specimens. Imaging system 100 can include anaccess means/member 110 to provide convenient, safe and light-tightaccess to sample environment 108, such as a door, opening, labyrinth,and the like. Additionally, sample environment 108 is preferably adaptedto provide atmospheric control for sample maintenance or soft x-raytransmission (e.g., temperature/humidity/alternative gases and thelike). Imaging system 100 can be a unitary system. Alternatively,imaging system 100 can be a modular unit adapted to be used or matedwith electronic imaging system such as electronic imaging system 10.

FIGS. 5-7 more particularly illustrate elements of sample object stage104 and an optical interface relative with the focal plane of camera andlens system 18. FIG. 5A shows a diagrammatic side view of sample objectstage 104 showing the relative movement of a movable phosphor plate 125according to the invention relative to the sample object stage. FIG. 5Bshows a diagrammatic side view of the sample object stage in a firstimaging position P1 wherein phosphor plate 125 is disposed proximate thesample object stage and positioned for imaging light from a phosphorlayer 132, as shown in FIG. 6. FIG. 5C shows a diagrammatic side view ofthe sample object stage in the second imaging position P2 whereinphosphor plate 125 has been withdrawn to a position that is notproximate the sample object stage. FIG. 6 shows an enlarged, fragmentarysectional view taken along line 6-6 of FIG. 5B, which corresponds withthe first imaging position P1. FIG. 7 shows an enlarged, fragmentarysectional view taken along line 7-7 of FIG. 5C, which corresponds withthe second imaging position P2.

Continuing with regard to FIGS. 6 and 7, sample object stage 104includes a support member made up from an open frame 120 to support andstretch a thin plastic support sheet 122. Support sheet 122 is selectedso as to support the weight of a sample or object to be imaged and ismade from a material that is optically clear and free of significantinterfering fluorescence. Phosphor plate 125 is mounted for motiontoward and away from sample object stage 104. While those skilled in theart might recognize other configurations, in a preferred embodiment,phosphor plate 125 is mounted for translation to provide slidable motion(in the direction of arrow A in FIG. 5A) relative to frame 120, beneaththe sample, in intimate contact with support sheet 122. Such motion canbe accomplished using methods known to those skilled in the art, forexample, frame 100 and phosphor plate 125 can be disposed on railssupported by a surface of an optical platen 126. As will be moreparticularly described below, in first imaging position P1, phosphorlayer 130 in phosphor plate 125 is in overlapping arrangement withsample object stage 104 (FIG. 6) when an x-ray image of the object iscaptured. In second imaging position P2, phosphor plate 125 istranslated or moved away from sample object stage 104 (FIG. 7) forcapture of an image of the object such that phosphor plate 125 is notimaged when an image of the object is captured in second imagingposition P2.

FIG. 6 provides an enlarged view of sample object stage 104 includingphosphor plate 125 to more particularly show a focal plane. Samplesupport sheet 122 preferably comprises Mylar or polycarbonate and has anominal thickness of about 0.1 mm. A protective layer 128 (for example,reflective Mylar) of about 0.025 mm is provided on phosphor layer 130 toprotect the surfaces of layer 130 during movement of phosphor plate 125.Protective layer 128 promotes or increases the image-forming lightoutput. In a preferred embodiment, protective layer 128 is reflective soas to prevent object reflection back into the image-forming screen,reducing confusing of the ionizing radiation image.

Phosphor layer 130 functions to transduce ionizing radiation to visiblelight practically managed by lens and camera system 18 (such as a CCDcamera). Phosphor layer 130 can have a thickness ranging from about 0.01mm to about 0.1 mm, depending upon the application (i.e., soft x-ray,gamma-ray or fast electron imaging). On the underside of phosphor layer130, as illustrated, an optical layer 132 is provided for conditioningemitted light from phosphor layer 130. Optical layer 132 can have athickness in the range of less than about 0.001 mm. Particularinformation about phosphor layer 130 and optical layer 132 are disclosedin U.S. Pat. No. 6,444,988 (Vizard), commonly assigned and incorporatedherein by reference. A supporting glass plate 134 is provided. Glassplate 134 is spaced at a suitable mechanical clearance from opticalplaten 126, for example, by an air gap or void 136. In one embodiment,the surfaces of clear optical media (e.g., a lower surface of glassplate 134 and both surfaces of optical platen 126) are subject toanti-reflective coating to minimize reflections that may confuse theimage of the object. FIG. 7 provides an expanded view of sample objectstage 13 including wherein phosphor plate 125 is removed (i.e., takenalong line 7-7 of FIG. 5C). Shown in FIG. 7 are frame 120, samplesupport sheet 122, an air gap/void 138 (since phosphor plate 125 isremoved), and optical platen 126.

Referring now to FIG. 8, in operation, in Step 200 an object (such as asmall animal) is immobilized on sample object stage 104. An operatorconfigures system 100 for imaging in a first mode, and in Step 202 animage of the object is captured using lens and camera system 18 in thefirst mode. System 18 converts the light image into an electronic imagewhich can be digitized. This digitized image is referred to as Image1 orI1. The digitized image can be displayed on the display device, storedin memory, transmitted to a remote location, processed to enhance theimage, and/or used to print a permanent copy of the image. The objectremains immobilized on sample object stage 104; no change in theposition/location of the object is made. The operator configures system100 for imaging in Step 204 and an image of the object is captured usinglens and camera system 18 in a second mode. The resulting digitizedimage is referred to as Image2 or I2. Since the position of the objectwas not moved or changed during the capture of the images, both Image1and Image2 can readily be merged or superimposed in Step 206, usingmethods known to those skilled in the art, such that the two images areco-registered. As such, a third image can be generated comprising Image1and Image2. In Step 208, the animal is removed from the object stage.

As indicated above, system 100 can be configured in several modes,including: x-ray imaging, bright-field imaging, dark-field imaging(including luminescence imaging, fluorescence imaging) and radioactiveisotope imaging. To configure system 100 for x-ray imaging or isotopeimaging, phosphor plate 125 is moved to position P1 in opticalregistration with sample object stage 104 (as shown in FIGS. 5B and 6).For an x-ray image, x-ray source 102 is employed when capturing theimage of the immobilized object. To configure system 100 forbright-field imaging or dark-field imaging (including luminescenceimaging and fluorescence imaging) without moving the immobilized objectand the support member or object stage, phosphor plate 125 is moved toposition P2, out of optical registration with sample object stage 104(as shown in FIGS. 5C and 7), and an image of the immobilized object isappropriately captured. The object is immobilized on sample object stage104, and light emitted from the object (usually diffusive within theturbid constituents of a solid object) is projected to the objectsurface, which intimately bears upon the upper surface of sample supportsheet 122.

For the purpose of optical imaging, the object surface is defined by arefractive boundary (e.g., the skin of an animal) that delineates theinterior of the object (usually a heterogeneous, turbid media of higherindex of refraction) and air. Light emanating from within an object(e.g., luminescent or transmitted) projects to the surface from which itscatters, defining the light that may be productively managed to createan image of the object. Conversely, light may be provided from beneathoptical platen 126 and scattered from the object surface, therebyproviding reflective light for imaging the same object. For opticalimaging, the definition of the object boundary may be moderated bymatching the refractive index of the object boundary to support sheet122 by introducing an index-matching fluid (e.g., water). The depth towhich good focus can be achieved in optical imaging is dependent onminimizing the surface scatter of the object, and methods such as indexmatching and increasing wavelength (e.g., near-infrared imaging) arewell known in the art.

The emitted sample light can arise from luminescence, fluorescence, orreflection, and the focal plane of the lens can be adjusted to theelevation of object surface. Alternatively, the “light” can be ionizingradiation passing through or emitted from the object, or passing intothe phosphor and forming an image. Soft x-rays, consistent with thinobjects or small animals, project an image through the diffusivephosphor onto the optical boundary, adding the depth of the (more thanabout 0.02 mm) to the depth of focus. More significant is the focaldistance contributed by the phosphor support plate 134, which may befractional millimeters, depending upon the thickness and index of theglass or plastic. The fractional-millimeter elevation of the best focalplane contributed by the phosphor support can provide a bettercoincidence between the phosphor focal plane and the focal plane usedfor optical imaging. For near-infrared optical imaging, thepreferred/best focal plane may be located at millimeter depths into anominally turbid object. The phosphor support plate 134 can be thickerto maximize the coincidence of the optical and phosphor imaging planes.Those skilled in the art will recognize how to tune the materials of thepresent invention to optimally co-locate the preferred optical andphosphor imaging planes. Currently described materials may bepractically assembled to assure multi-modal focal plane co-location toaccommodate the demands of a fast lens system.

Appropriately fast lens systems for dark-field and x-ray imagingapplications will likely have sub-millimeter focal depths, necessitatingthe above considerations. Accordingly, for a particular embodiment, itmay be desirable for multiple optical elements to enable the location ofa common focal plane shared by differing modes of imaging.

Emitted gamma rays from a thick object (such as 99Tc emission from ananimal organ) are distributed over the plane of the phosphor, diffusingthe image by millimeters, and an appropriately thick phosphor layer(about 0.1 mm) may be preferred for increased detection efficiency.Consequently, the location of the focal plane at the supporting sheet isnot critical to the resolution of the radio isotopic image. Betterresolution and more precise planar projection of the emitting isotopecan be achieved by gamma-ray collimation. Collimators ofmillimeter-resolution are available and capable of projecting isotopiclocation to millimeter resolution at the focal plane of the phosphor inthe present invention.

Of particular relevance to the operation of the present invention is thethickness of the layers in the focal plane of the lens. For example,fast lenses, (which are essential elements for the practice of imaginglow-light emissions) will have a focal depth of focus of about 0.5 mmfor very fast lenses. For good resolution of objects of interest, lessthan about 0.2 mm of spatial resolution is desirable, and a megapixelCCD camera (cooled) imaging at 100 mm field is suitable. Generally, moreresolution is desirable.

Precision registration of the multi-modal image can be accomplishedusing methods known to those skilled in the art. By placing the objecton a thin, stretched optical support that allows phosphor plate 125 tobe removed without displacement of the object, co-registered opticalimaging is enabled by the same lens and camera system usingepi-illumination methodologies at a sufficiently similar focal plane.

Examples are now provided. FIGS. 9A-9C show images captured using theapparatus and the method of the present invention. A mouse wasimmobilized on sample object stage 104 (step 200 of FIG. 8) of system100. System 100 was first configured for near-infrared fluorescenceimaging wherein phosphor plate 125 is removed from co-registration withframe 100. A first image was captured and is displayed in FIG. 9A (step202 of FIG. 8). Next, system 100 was configured for x-ray imagingwherein phosphor plate 125 is placed in co-registration with frame 100.A second image was captured and is displayed in FIG. 9B (step 204 ofFIG. 8). Using methods known to those skilled in the art, the image ofFIG. 9A was thresholded to make transparent those regions with pixelintensity values less than the threshold value and then the thresholdedimage was merged with the image of FIG. 9B in step 206 of FIG. 8; andthe merged image is displayed in FIG. 9C. Note that the fluorescentsignals superimposed on the anatomical reference clarify the assignmentof signal to the bladder and an expected tumor in the neck area of thisillustrated experimental mouse. It is noted that the first and/or secondimage can be enhanced using known image processing methods/means priorto be merged together. Alternatively, the merged image can be enhancedusing known image processing methods/means. Often, false color is usedto distinguish fluorescent signal from gray-scale x-rays in a mergedimage.

FIGS. 10A-10C provide a further example using an apparatus suitable foruse in accordance with the method of the present invention. FIG. 10A isa near-infrared fluorescence image of a mouse while FIG. 10B is an x-rayimage of the same immobilized mouse. Using methods known to thoseskilled in the art, the first and second images were merged in themanner previously described and the merged image is displayed in FIG.10C. Prior to being merged, the first and second images were contrasted,using methods known to those skilled in the art. This processing allowsparticular areas of the mouse to be visually enhanced for diagnosticpurposes. For example, areas 150, 152, and 156 are potential secondaryearly detection sites, and area 154 shows the primary tumor injectionsite at the knee.

FIGS. 11A-11C provide yet a further example using an apparatus suitablefor use in accordance with the method of the present invention. FIG. 11Ais a near-infrared fluorescence image of a mouse wrist while FIG. 11B isan x-ray image of the same immobilized mouse wrist. Using methods knownto those skilled in the art, the first and second images were merged inthe manner previously described and the merged image is displayed inFIG. 11C. The merged image provides a diagnostic image for viewing apotential secondary tumor site. Note that this image set clearlydemonstrates the precision with which the apparatus of FIGS. 1 to 8enables the co-location of images of objects from differing imagingmodes. The maximum fluorescent signal emanating from a pre-metastatictumor on the radius (arm-bone) tip at the wrist is within about 0.1 mmof the suspect lesion subsequently identified by microscopic histology.

A phosphor plate suitable for use with the method of the presentinvention is disclosed in U.S. Pat. No. 6,444,988 (Vizard), commonlyassigned and incorporated herein by reference. A phosphor plate asdescribed in Vizard is shown in FIG. 12. A suitable phosphor plate 125Afor use with the apparatus and the method of the present inventionincludes a transparent support 210 (such as glass) upon which is coatedan interference filter 220 which is a multicoated short-pass filterdesigned to transmit light at a specified wavelength (and below) andreflect light above that wavelength. Plate 125A also includes a thinphosphor layer 240 and a removable thick phosphor layer 260. Thinphosphor layer 240 is used for high resolution imaging applications ofionizing radiation or for very low energy (self-attenuating) ionizingradiation such as low-energy electrons or beta particles. Thick phosphorlayer 260 is used for high-energy ionizing radiation that freelypenetrates the phosphor. Thick phosphor layer 260 is removable and isshown in FIG. 12 overlaying thin phosphor layer 240. Layer 260 isremovable to the position shown in dashed lines out of contact withlayer 240.

The phosphor preferably used in phosphor layers 240 and 260 isGadolinium Oxysulfide Terbium whose strong monochromatic line output(544-548 nanometers (NM) is ideal for co-application with interferenceoptics. This phosphor has technical superiority regarding linear dynamicrange of output, sufficiently “live” or prompt emission and timereciprocity, and intrascenic dynamic range which exceed other phosphorsand capture media. This phosphor layer preferably has a nominalthickness of 10-30 micrometers (μm) at 5-20 grams/square foot (g/ft²) ofphosphor coverage, optimally absorbing 10-30 Kev x-rays. Thick phosphorlayer 260 has a nominal thickness of 100 μM at 80 g/ft² of phosphorcoverage.

The duplex phosphor layers impart flexibility of usage for which thethick phosphor layer 260 may be removed to enhance the spatialresolution of the image. Thin phosphor layer 240 intimately contactsfilter 220, whereas thick phosphor layer 260 may be alternatively placedon thin phosphor layer 240. Interference filter 220 transmits light at551 NM and below and reflects light above that wavelength. Filter 220comprises layers of Zinc Sulfide-Cryolite that exhibits a largereduction in cutoff wavelength with increasing angle of incidence. Thefilter has a high transmission at 540-551 NM to assure good transmissionof 540-548 NM transmission of the GOS phosphor. The filter also has asharp short-pass cut-off at about 553 NM, that blue shifts at about 0.6NM per angular degree of incidence to optimize optical gain. Glasssupport 210 should be reasonably flat, clear, and free of severedefects. The thickness of support 210 can be 2 millimeters. The oppositeside 280 of glass support 210 is coated with an anti-reflective layer(such as Magnesium Fluoride, green optimized) to increase transmittanceand reduce optical artifacts to ensure that the large dynamic range ofthe phosphor emittance is captured.

Referring now to FIG. 13, there is shown a method of producing phosphorlayer 240. In Step 300, a mixture of GOS:Tb in a binder is coated on apolytetrafluoroethylene (PTFE) support. The PTFE support enables releaseof the coated phosphor layer from the PTFE support and subsequent use ofthe phosphor layer without support, since conventional supportingmaterials are an optical burden to screen performance. For the thinphosphor layer 240, in Step 320 an ultra thin (about 0.5 g/ft², 0.5 μmthick) layer of cellulose acetate overcoat can be applied to offerimproved handling characteristics of the thin phosphor layer and toprovide greater environmental protection to the underlying opticalfilter. In Step 340, the phosphor layer is removed from the PFTEsupport. The thin phosphor layer overcoated side is overlayed oninterference filter 220 in Step 360. Clean assembly of the thin phosphorlayer 240 and filter 220 assures an optical boundary that optimizesmanagement of screen light output into the camera of the lens/camerasystem. Optical coupling of layer 240 and filter 220 is not necessary,since performance reduction may result. In Step 380, layer 240 can besealed around its periphery and around the periphery of filter 220 formechanical stability and further protection of the critical opticalboundary against environmental (e.g., moisture) intrusion.

Advantages of the method of the present invention include: anatomicallocalization of molecular imaging agent signals in small animals,organs, and tissues; precise co-registration of anatomical x-ray imageswith optical molecular and radio isotopic images using one system;improved understanding of imaging agent's biodistribution throughcombined use of time lapse molecular imaging with x-ray imaging; andsimple switching between multi-wavelength fluorescence, luminescence,radio-isotopic, and x-ray imaging modalities without moving theobject/sample.

Reference is made to the following commonly assigned, copending U.S.patent applications: Ser. No. 12/381,599 filed Mar. 13, 2009 by Feke etal, entitled METHOD FOR REPRODUCING THE SPATIAL ORIENTATION OF ANIMMOBILIZED SUBJECT IN A MULTI-MODAL IMAGING SYSTEM; and Ser. No.12/475,623 filed Jun. 1, 2009 by Feke et al, entitled TORSIONAL SUPPORTAPPARATUS AND METHOD FOR CRANIOCAUDAL ROTATION OF ANIMALS, thedisclosures of both of which are incorporated by reference into thisspecification.

FIGS. 14A, 14B, 15A and 15B show a diagrammatic partial view of thesample stage 104 (a transparent tube in this instance) of the imagingsystem 100 of FIGS. 3A and 3B where the subject mouse 112 is positionedin a chamber 113 and administered immobilizing anesthesia through arespiratory device 114 connected to an outside source via a tube 115which enters the sample environment 108 via the light-locked gas ports.A rotational mechanism 116 may be provided for adjusting the rotationalposition of the mouse about its craniocaudal axis. A translationalmechanism 117 may be provided to adjust the axial location of the mouserelative to source 102 and fiber optics 106. Further details of thestructure shown in these figures are disclosed in the first applicationof Feke et al, mentioned in the preceding paragraph. An X-ray anatomicalimage 140 and a near-infrared fluorescence image 142 are acquired of thegastro-intestinal tract of the immobilized subject mouse 112. Images140, 142 are shown side by side in FIGS. 16A and 16B.

In a preferred embodiment of the present invention, referring to FIGS.14A and 14B, an X-ray image 140 provides a convenient anatomical map ofmusculoskeletal features of the subject mouse 112. The X-ray image 140is an orthogonal imaging modality that will not pollute optical signalsemanating from the subject. While the skeleton provides the framework tospatially assign organs, X-ray contrast agents may be administered tothe mouse in accordance with the invention to provide an effective meansto delineate tissues that typically give poor contrast by X-ray.

FIG. 21 shows an anatomical X-ray image of a mouse in the sample chamberon the sample object stage of the imaging system of FIGS. 3A and 1B.Labels refer to regions A: Bones/Joints, B: Heart, C: Lungs, D: Liver,E: Kidneys, F: GI Tract, and G: Bladder. Since bones are densestructures they absorb X-rays and appear dark. One can immediately notethe fine structure in bones that are sub-millimeter in scale, like therib cage and fibia bones in the leg. Many disease models involving theskeleton may be non-invasively studied using such images. These includebone growth and damage in response to environmental or physical inputs.Furthermore, subtle changes in bone density may also be measured. Whilebones are dense and provide positive contrast that appears black, air isobviously of low density and absorbs very little X-ray radiation. Thus,locations in an animal that contain gases give contrast toward the whiteend of the intensity spectrum shown in FIG. 21. One area in which air isplentiful is in the lungs (FIG. 21 region C). In fact, the rib cage isoften considered a cavern of air due to the presence of the lungs, whichappear as a triangular pattern on each half of the rib cage.Incidentally, another area in which gas tends to build up and givenegative contrast on X-ray is in the bowels (FIG. 21 region F).Nevertheless, the air cavern of the rib cage is home to anotherimportant organ: the heart (FIG. 21 region B). Since the tissuecomprising the heart has higher density than air, it gives positive(dark) contrast in comparison to the lungs. Indeed, since this organ iseffectively surrounded by air from the lungs, it is often described as a“heart shadow” in the rib cage. The size of the heart shadow may bemeasured as it expands into the lung area. If its size increases toomuch, it is an indication of pulmonary edema, a condition in which theheart fills with liquid and expands to unsafe levels. Pulmonary edemahas been measured and studied in rat models of heart disease. While theheart and lungs are soft tissue systems that are readily observed byX-ray, other organs possess little innate contrast to permit theirimaging and require an alternate strategy. Organs like the stomach,gastrointestinal tract, kidneys, bladder and liver give little to nocontrast during X-ray imaging. FIG. 21 regions D-F shows the lack ofdiscernable contrast from each of these various tissue systems in theabdomen. However, through the use of x-ray contrast agents in accordancewith the present invention, these organs can be delineated and studiedin a non-invasive fashion using the X-ray modality. Molecules thatincorporate atoms with exceptional X-ray absorption properties aretypically utilized as contrast agents. Barium and iodine meet thiscriterion and are two of the most widely used atomic components of X-raycontrast agents. Indeed, both of these reagents have been utilized inthe clinic for decades to perform gastric and heart perfusion imaging,among others. X-ray contrast agents effectively absorb X-rayirradiation, thus providing contrast in the organs in which they reside.Different X-ray contrast agents may be chosen depending on the organ tobe imaged. For example, barium sulfate may be administered orally to thesubject to view its stomach and gastrointestinal tract as shown inimages 140 and 142 of FIGS. 14B and 16A. Iodinated contrast agents maybe used intravenously to image the kidneys of the animal under study asshown in images 144 and 146 of FIG. 17. Other agents like goldnanoparticles may be used to add X-ray contrast to the liver, kidneys,or tumors.

The x-ray contrast agent may be administered at various time points inan imaging study to assign and delineate organs using X-ray imaging. Aresearcher will use that anatomical information to determine thecontribution of fluorescent or luminescent signal emanating from thatorgan at that time point. For example as shown by the images in FIGS.17A, 17B and 17C, a researcher may administer a fluorescent probe intoan animal at time zero. At this and subsequent time points, the X-raycontrast agent may be given to the subject. A fluorescence image 146 isthen acquired to capture signal from the original fluorescence probe asshown in FIG. 17B, and is immediately followed by an X-ray image 144 toview the organs as shown in FIG. 17A. In a final step, the images arethen overlaid, or combined, to determine co-localization of thefluorescent signal to the organ of interest as shown in image 148 ofFIG. 17C. Alternatively, the fluorescence imaging probe and the x-raycontrast agent may be administered to the mouse essentiallysimultaneously.

Referring now the workflow shown in FIG. 18 and FIGS. 17, 19 and 20, inStep 400 the optical imaging agent and X-ray contrast agent areintroduced into the subject 112 as per the experimenter's protocol. InStep 410, the subject mouse 112 is placed on the object stage 104. Theanatomical X-ray image 144 and near-infrared fluorescence image 146 areacquired in Step 420 and a co-registered image 148 of the anatomicalX-ray image 144 and near-infrared fluorescence image 146 shown FIG. 17is created in Step 430.

An example of this strategy in practice is given in FIGS. 14A through16B, previously discussed. In this case, the experimenter is attemptingto localize a fluorescent signal with the gastrointestinal (GI) tract.Since this sizable organ yields poor contrast in an X-ray image, anx-ray contrast agent must be utilized. Agents such as barium sulfatehave been used in the clinic for decades to resolve GI features. Sincebarium salts are generally insoluble and inert, they may be safely usedin the human GI tract, either orally or rectally, to provide contrastduring X-ray imaging. After imaging is complete, the barium is excretedfrom the subject without absorption into the body. However, the use ofagents such as barium sulfate in small animal imaging has been minimal,and they have not been used in a multimodal approach to localizefluorescent or luminescent signals. The images shown in FIGS. 14B, 15B,16A and 16B illustrate how contrast agents like barium sulfate may beutilized for the anatomical co-registration of fluorescent orluminescent signals during optical imaging. These figures show anexample of a mouse after one hour of consumption of 40 mg of a 1:1mixture of barium sulfate and creamy peanut butter combined with KodakX-Sight 761 Nanospheres, which are near-infrared fluorescentnanoparticles (commercially available from Carestream Health, Inc.). Inthis case, the contrast agent and fluorescent nanoparticles wereadministered simultaneously. Subsequent experiments could possibly usethe barium sulfate/peanut butter mix alone to determine the clearance ofthe original dose of fluorophore. FIGS. 14B and 16A show the X-ray image140 with excellent barium sulfate contrast (Target/Non-Target=1.33) ofthe GI tract. The near-infrared fluorescence image 142 of FIGS. 15B and16B show near-infrared fluorescence from the nanoparticles. In thiscase, the origin of the optical signal is coincident with barium sulfatecontrast of the X-ray image 140.

In addition to barium sulfate, iodinated contrast agents as well as goldnanoparticles may be used as X-ray contrast agents for various tissues.Iodine is a synthetically accessible atom with sufficient electrondensity to yield X-ray contrast. Thus, it has been incorporated intoseveral compounds that may be synthesized as water soluble throughcontrast agents. These reagents are generally used for the purposes ofintravenous injection since they are not harmful, and will rinse out ofthe subject through the renal pathway. Several iodine based contrastagents are commercially available for use in humans for imaging of heartvasculature and other bulk tissues. FIGS. 17A to 17C, 19A and 19B, and20A and 20B show the utilization one such agent, Visipaque™ (iodixanol)commercially available from GE Healthcare, to localize a fluorescentsignal in the renal system of mice. The mouse was administered anintravenous injection of 200 μL of Visipaque (iodixanol) (320 mg/mliodine) combined with Kodak X-Sight 670 Large Stokes Shift dye, which isa near-infrared fluorescent dye (commercially available from CarestreamHealth, Inc.). The contrast agent and dye concentrated in the kidneysthereby providing clear contrast in them.

FIG. 22 shows a graph of the excess x-ray density in the medullaryregions of the kidneys conferred by the Visipaque vs. the injectedvolume of the Visipaque. Varying volumes of Visipaque were mixed withcomplementarily varying volumes of phosphate buffered saline to achievea series of 200 μL total injection volumes. Mice were immobilized, andX-ray images were acquired both before and 10 minutes after injection ofthe different Visipaque injection volume for each mouse. The imagesbefore injection were subtracted from the images after injectioncorresponding to each mouse receiving a different Visipaque injectionvolume to create difference images, and a region-of-interest analysiswas performed for the medullary regions of the kidneys in the differenceimages to measure the excess x-ray density conferred by the Visipaque inthe medullary regions. The graph shows that the excess x-ray density inthe medullary regions of the kidneys, wherein the excess x-ray densityof both kidneys was averaged together for each mouse, is directlyproportional to the injected volume of the Visipaque, with a constant ofproportionality of 0.0032 as determined by a linear fit of the data.

As indicated earlier in this specification, the x-ray contrast agentsand optical imaging agents may be targeted, non-targeted, or both. Inaccordance with the invention, the x-ray contrast agent may be targetedwhile the optical imaging agent is non-targeted; or the x-ray contrastagent may be non-targeted while the optical imaging agent is targeted;or both agents may be targeted; or both agents may be non-targeted. Thefollowing table lists the agents previously mentioned along with otherknown agents that the inventors consider appropriate for use in anyconvenient combination suited for an anatomical region of interest,without departing from the scope of the present invention.

Targeted or Anatomical Agent Modality Non-targeted Regions CommentsBarium X-ray Non-targeted Gastrointestinal Example in sulfate tract thisspec Barium X-ray Non-targeted Vasculature sulfate in gelatin (Baritop)Iodinated X-ray Non-targeted Vasculature, Example in contrast renal thisspec agents Fenestra VC X-ray Non-targeted Vasculature, renal FenestraLC X-ray Non-targeted Hepatobiliary eXIA 160 X-ray Targeted VasculatureRadiocontrast eXIA 160 X-ray Non-targeted Hepatobiliary Radiocontrastand Splenic Gold X-ray Non-targeted Vasculature, nanoparticles renal,tumors Corrosion X-ray Non-targeted Vasculature casting materialBatson's 17 with added lead pigment Silicon X-ray Non-targetedVasculature rubber; Microfil MV122 Agents X-ray Targeted Variousdisclosed in U.S. Pat. No. 5,141,739 Kodak X- Optical Non-targetedGastrointestinal Example in Sight tract this spec Nanospheres Kodak X-Optical Non-targeted Vasculature, Example in Sight hepatobiliary thisspec Nanospheres Kodak X- Optical Non-targeted Renal Example in Sightlarge this spec Stokes shift dyes Gold X-ray Non-targeted Vasculature,nanoparticles renal, tumors Corrosion X-ray Non-targeted Vasculaturecasting material Batson's 17 with added lead pigment Silicon X-rayNon-targeted Vasculature rubber; Microfil MV122 Agents X-ray TargetedVarious disclosed in U.S. Pat. No. 5,141,739 Kodak X- OpticalNon-targeted Gastrointestinal Example in Sight tract this specNanospheres Kodak X- Optical Non-targeted Vasculature, Example in Sighthepatobiliary this spec Nanospheres Kodak X- Optical Non-targeted RenalExample in Sight large this spec Stokes shift dyes Kodak X- OpticalTargeted Various Sight Nanosphere Conjugates Kodak X- Optical TargetedVarious Sight large Stokes shift dye conjugates Qdots OpticalNon-targeted Qdot Optical Targeted Vascular, conjugates hepatobiliaryVarious dyes Optical Non-targeted Various Various dye Optical TargetedVascular conjugates Fluorescent Optical Non-targeted Vasculature, silicarenal, hepatobiliary Fluorescent Optical Targeted Various silicaconjugates Nanoparticles Optical Non-targeted Vascular, derived fromhepatobiliary self-assembly of amphiphilic copolymers NanoparticlesOptical Targeted Various derived from self-assembly of amphiphiliccopolymers Fluorescent Optical Targeted Various proteins LuciferaseOptical Targeted Various

The X-ray contrast agents described herein, when used as disclosed,provide a facile methodology for the anatomical co-registration of bothtargeted and non-targeted fluorescent and luminescent signals duringmolecular imaging.

The invention has been described in detail with particular reference toa presently preferred embodiment, but it will be understood thatvariations and modifications can be effected within the spirit and scopeof the invention. The presently disclosed embodiments are thereforeconsidered in all respects to be illustrative and not restrictive. Thescope of the invention is indicated by the appended claims, and allchanges that come within the meaning and range of equivalents thereofare intended to be embraced therein.

PARTS LIST

-   10 electronic imaging system-   12 light source-   14 optical compartment-   16 mirror-   18 lens and camera system-   20 communication and computer control system-   22 computer monitor-   100 imaging system of the present invention-   102 x-ray source-   104 sample object stage or support member-   106 epi-illumination; fiber optics-   108 sample environment-   110 access means/member-   112 subject mouse-   113 chamber for mouse 112-   114 respiratory device-   115 tube-   116 rotational mechanism-   117 translational mechanism-   120 frame-   122 support sheet-   125, 125A phosphor plate-   126 optical platen-   128 protective layer-   130 phosphor layer-   132 optical layer-   134 support plate; glass-   136 air gap or void-   138 air gap or void-   140 x-ray image-   142 near-infrared fluorescence image-   144 x-ray image-   146 near-infrared fluorescence image-   148 co-registered image-   150, 152, 156 potential secondary early detection sites-   154 primary tumor injection site-   200 to 208 method steps-   210 transparent support-   220 interference filter-   240 thin phosphor layer-   260 thick phosphor layer-   280 opposite side-   300 to 380 method steps-   400 to 430 method steps

1. A method of imaging a subject animal, comprising: treating the animalwith an x-ray contrast agent and an optical imaging agent; supportingthe animal in an immobilized state on a support member; providing aphosphor plate adapted to be disposed proximate the support member whencapturing a first image; with the phosphor plate disposed proximate thesupport member, imaging the immobilized animal in a first imaging modeto capture the first image, the first imaging mode being an x-ray mode;removing the phosphor plate from proximate the support member, aftercapturing the first image and while the object remains immobilized onthe support member; and with the phosphor plate removed from proximatethe support member, imaging the immobilized animal in a second imagingmode to capture a second image, the second imaging mode being an opticaldark-field mode.
 2. The method of claim 1, further comprising the stepof generating a third image by merging the first and second images,whereby features of the second image can be observed in relation tofeatures of the first image.
 3. The method of claim 1, wherein the x-raycontrast agent and the optical imaging agent are administeredsimultaneously to the animal.
 4. The method of claim 1, wherein thex-ray contrast agent and the optical imaging agent are administeredsequentially to the animal.
 5. The method of claim 1, wherein theoptical, dark-field image is a fluorescence image.
 6. The method ofclaim 1, wherein the x-ray anatomical image and the optical, dark-fieldimage are acquired using a common, shared focal plane.
 7. The method ofclaim 1, wherein the x-ray contrast agent is targeted and the opticalimaging agent is non-targeted.
 8. The method of claim 1, wherein thex-ray contrast agent is non-targeted and the optical imaging agent istargeted.
 9. The method of claim 1, wherein both the x-ray contrastagent and the optical imaging agent are targeted.
 10. The method ofclaim 1, wherein both the x-ray contrast agent and the optical imagingagent are non-targeted.
 11. A method of imaging a subject animal,comprising: treating the animal with an x-ray contrast agent and anoptical imaging agent; supporting the animal in an immobilized state ona support member; providing a phosphor plate movable relative to thesupport member, while the object remains immobilized on the supportmember, between a first position wherein the phosphor plate is inoptical registration with the support member and a second positionwherein the phosphor plate is not in optical registration with thesupport member; capturing an x-ray image of the immobilized animal whenthe phosphor plate in disposed in the first position; and capturing anoptical dark-field image of the immobilized animal when the phosphorplate in disposed in the second position.
 12. The method of claim 11,further comprising: generating a third image by merging the first andsecond images, whereby features of the second image can be observed inrelation to features of the first image; and displaying, transmitting,processing, or printing, the third image.
 13. The method of claim 11,wherein the x-ray anatomical image and the optical, dark-field image areacquired using a common, shared focal plane.
 14. An apparatus forimaging a subject animal, comprising: first imaging means for imagingsuch an animal in a first imaging mode to capture a first image, thefirst imaging mode being selected from the group: x-ray mode and radioisotope mode; second imaging means for imaging such an animal in asecond imaging mode that uses light from the immobilized animal tocapture a second image, the second imaging mode being selected from thegroup: bright-field imaging mode and dark-field imaging mode; a supportstage, fixedly mounted in the apparatus, for receiving such an animal inan immobilized state such that the animal is immobilized in theapparatus during imaging by the first and second imaging means withoutmovement of the animal from the support stage; and a movable phosphorplate to transduce ionizing radiation from the first imaging means tovisible light, the phosphor plate being mounted to be moved, while theobject remains immobilized on the support member, between a firstposition proximate the support stage during capture of the first imageand a second position not proximate the support stage during capture ofthe second image.
 15. A method for imaging a subject animal, comprising:providing a fixed support stage; receiving the animal on the supportstage in an immobilized state; imaging the immobilized animal on thesupport stage in a first imaging mode to capture a first image, thefirst imaging mode being selected from the group: x-ray mode and radioisotope mode; without moving the animal or the support stage, imagingthe animal on the support stage in a second imaging mode that uses lightfrom the immobilized animal to capture a second image, the secondimaging mode being selected from the group: bright-field imaging modeand dark-field imaging mode; providing a movable phosphor plate totransduce ionizing radiation from the first imaging means to visiblelight; and moving the phosphor plate, while the object remainsimmobilized on the support member, between a first position proximatethe support stage during capture of the first image and a secondposition not proximate the support stage during capture of the secondimage.
 16. The method according to claim 15, further comprising:treating the animal with an x-ray contrast agent and an optical imagingagent; and registering the first image with the second image, wherebyfeatures of the second image may be observed in relation to features ofthe first image.
 17. A method of imaging a subject animal, comprising:supporting the animal in an immobilized state on a support member;providing a phosphor plate movable relative to the support member, whilethe object remains immobilized on the support member, between a firstposition wherein the phosphor plate is in optical registration with thesupport member and a second position wherein the phosphor plate is notin optical registration with the support member; capturing a first imageof the immobilized animal when the phosphor plate in disposed in thefirst position, the first image being captured using an imaging modeselected from the group: x-ray mode and radio isotope mode; andcapturing a second image of the immobilized animal when the phosphorplate in disposed in the second position, the second image beingcaptured using an imaging mode selected from the group: bright-fieldimaging mode and dark-field imaging mode.